Polyphosphoinositide-derived diacylglycerol stimulates the hydrolysis of phosphatidylcholine by phospholipase C during exocytosis of the ram sperm acrosome. Effect is not mediated by protein kinase C.

Abstract

In ram spermatozoa, treatment with the ionophore A23187 and Ca2+ led to an increase in total diacylglycerol mass and to exocytosis of the acrosomal granule. If sperm cells were prelabeled with [3H]palmitic acid, stimulation with A23187/Ca2+ resulted in the generation of [3H]diacylglycerols with a mixture of saturated and unsaturated fatty acids. When cells were prelabeled with 1-O-[3H]octadecylglycerophosphocholine, stimulation led to the generation of [3H]alkylacylglycerol. No rise in [3H]diacyl- or [3H]alkylacylphosphatidic acid was detected under these conditions. Moreover, no changes in the mass of phosphatidic acid have been previously noted under similar conditions. Thus, these results indicate that diradylglycerols are generated via phospholipase C (PLC). Increases in diradylglycerols were paralleled by rises in monoacyl- or monoalkylglycerols labeled at position 1, but not in free [3H]palmitic acid or [3H]octadecanol, implying that, unlike somatic cells, spermatozoa catabolize diradylglycerols via a 2-diglyceride lipase. Activation of PLC appears to be effected by phosphoinositide-derived diacylglycerol: exposure to Mg2+, a cation known to inhibit phosphoinositide hydrolysis, resulted in less PLC activity upon stimulation, and addition of exogenous 1,2-diacylglycerols enhanced the enzyme's activity. However, 1,3-diacylglycerol and alkylacylglycerol also stimulated PLC activity, suggesting that the effect is unlikely to be mediated via protein kinase C. Since diradylglycerols are known to be essential in the molecular sequence leading to membrane fusion in mammalian spermatozoa, these results suggest that their generation via PLC constitutes a fundamental event during acrosomal exocytosis in response to physiological agonists.